The human immunodeficiency virus type 1 (HIV-1) transactivator Tat protein is essential for efficient viral gene expression and virus replication. We have previously shown that Tat interacts with the basal transcription factor TFIID. This interaction is mediated through the amino acid 36-50 core domain of Tat. We now demonstrate that soluble peptide analogs of the Tat core domain are able to effectively block LTR transactivation. In cotransfection experiments, Tat peptide analogs inhibited Tat transactivation of an HIV-1 LTR-CAT reporter construct up to 80-fold. In contrast, inhibition of other promoters such as HTLV-I and CMV was approximately two-fold. One of the Tat peptide analogs, containing amino acid substitutions at position 41 and 44, inhibited HIV virus replication by 85 percent in latently infected U1 cells induced with Tat. While both short and long peptide analogs (amino acids 36-50 vs 36-72) inhibited Tat transactivation in transient assays, the short peptides were more effective inhibitors of virus replication in U1 cells. Furthermore, U1 cells treated with the Tat peptide 36-50 (41/44) analog showed markedly delayed virus transmission when co-cultivated with parental U937 cells. The Tat peptide analog did not decrease expression of cellular genes including -actin, GAPDH and histone H2b. Finally, it is demonstrated that the Tat peptide analog inhibits Tat transactivation through a TBP-activated pathway using a minimal HIV promoter.